Figure 4

Southern blot mapping of nucleosomes on the episomal pX-tSL cassette. A) Unlike the results for the genomic array, the pX-tSL cassette shows no preferential protection. The letter below each blot refers to the specific oligonucleotide hybridization probe, as listed in Table 1. The probe location is indicated by the lines above the schematic. The schematic shows the location of the SL RNA gene, the upstream promoter elements, and the borders of the cassette (thick black lines). The tag sequence is represented as a white box. The histogram represents the ratios of the genomic signal:monosome signal. Lane1, Sac II digest of mock-treated DNA; lane 2, Bst EII/Not I digest of the pX-tSL mock-treated DNA; lane 3, MNase-digested chromatin. B) Three hypothetical episomal nucleosome array conformations, to which only the first is able to bind the SL RNA gene pre-initiation complex. Assuming the periodicity of the nucleosomes on the drug-selectable marker-carrying episome are regular for L. tarentolae, the tSL RNA gene promoter has at most a 26% chance of exposure to cognate transcription factors.